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Combining Yeast Display and Competitive FACS to Select Rare Hapten-Specific Clones from Recombinant Antibody Libraries

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      Abstract

      The development of antibodies to low molecular weight haptens remains challenging due to both the low immunogenicity of many haptens and the cross-reactivity of the protein carriers used to generate the immune response. Recombinant antibodies and novel display technologies have greatly advanced antibody development; however, new techniques are still required to select rare hapten-specific antibodies from large recombinant libraries. In the present study, we used a combination of phage and yeast display to screen an immune antibody library (size, 4.4 × 10 6) against hapten markers for petroleum contamination (phenanthrene and methylphenanthrenes). Selection via phage display was used first to enrich the library between 20- and 100-fold for clones that bound to phenanthrene–protein conjugates. The enriched libraries were subsequently transferred to a yeast display system and a newly developed competitive FACS procedure was employed to select rare hapten-specific clones. Competitive FACS increased the frequency of hapten-specific scFvs in our yeast-displayed scFvs from 0.025 to 0.005% in the original library to between 13 and 35% in selected pools. The presence of hapten-specific scFvs was confirmed by competitive ELISA using periplasmic protein. Three distinct antibody clones that recognize phenanthrene and methylphenanthrenes were selected, and their distinctive binding properties were characterized. To our knowledge, these are first antibodies that can distinguish between methylated (petrogenic) versus unmethylated (pyrogenic) phenanthrenes; such antibodies will be useful in detecting the sources of environmental contamination. This selection method could be generally adopted in the selection of other hapten-specific recombinant antibodies.

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            Author and article information

            Affiliations
            []Department of Biochemistry and Molecular Biology, Tulane University School of Medicine , New Orleans, Louisiana, United States
            [§ ]Bioscience Division, Los Alamos National Laboratory , Los Alamos, New Mexico, United States
            []Department of Pathology, University of Texas Medical Branch , Galveston, Texas, United States
            Author notes
            Journal
            Anal Chem
            Anal. Chem
            ac
            ancham
            Analytical Chemistry
            American Chemical Society
            0003-2700
            1520-6882
            29 August 2016
            20 September 2016
            : 88
            : 18
            : 9181-9189
            27571429 5032104 10.1021/acs.analchem.6b02334
            Copyright © 2016 American Chemical Society

            This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

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            Custom metadata
            ac6b02334
            ac-2016-023345

            Analytical chemistry

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